There exists much current effort and research being expended toward the genetic transformation of plant species. It is believed that the development of efficient means for transforming foreign genes into plant germ lines will allow the diversity of the genetic stock in commercially important crop species to be widened and to allow functional genes of specific interest to be selectively introduced into crop species. The effort and research to date on the transformation, or genetic engineering, of plant species has achieved results which vary quite dramatically depending on the species of plant.
The principal mechanism which has been used heretofore for the introduction of exogenous genes into plants has begun with the transformation of single plant cells, either as protoplasts, or in an undifferentiated tissue mass known as a callus. Chimeric genes functional in plant cells have been introduced into single cell plant protoplasts by electroporation and microinjection. However, the most widely used transformation technique to date has taken advantage of a natural trait of the plant pathogen Agrobacterium tumefaciens, which has the innate ability to transfer a portion of the DNA from a Ti (Tumor-inducing) plasmid harbored in it into an infected plant cell. By inserting foreign genes into plasmids in Agrobacterium which carry certain sequences from the Ti plasmid, the bacterial transformational trait can be used to transport the foreign genes into the genome of the infected plant cells. Agrobacterium-mediated plant cell transformation has been found to work reasonably well in many model crop species, such as tobacco, petunia and carrot, but does suffer from several significant limitations. The first limitation is that the transformation can only be done on a tissue culture level, typically with somatic tissues, which then must be regenerated artificially into a whole plant. This limits the applicability of Agrobacterium-mediated genetic transformation to those crop species which can readily be regenerated from types of tissues which are susceptible to Agrobacterium infection. This limitation can also make Agrobacterium-mediated transformation a laborious process since the regeneration of some plants, even though possible, can be a long labor-intensive process requiring much skill and often some art. A second limitation is that the natural host range of Agrobacterium includes only dicotyledonous plants and a limited number of monocot species of the Liliaceae family. Therefore Agrobacterium-mediated transformation has not been proven to be an effective tool for monocot species of commercial interest, such as the cereal crop species. Another difficulty with Agrobacterium-mediated transformations is the generation of somoclonal variants, which spontaneously arise in plant tissues in tissue culture, which may complicate identification of transformants.
It has been demonstrated that at least some chimeric gene constructions are effective for expression of foreign genes in many popular crop plant cells. The functionality of these chimeric constructions in monocots as well as dicots has been demonstrated by the transformation of maize as well as soybean protoplasts in culture through such techniques as electroporation. Christou et al., Proc. Natl. Acad. Sci., USA, 84:3962-3966 (1987). However, no currently known methodology exists to regenerate whole soybean plants, or whole fertile plants of several other important crop species, from such protoplasts. No whole, intact transformed soybean plants, for example, are known to have been regenerated from protoplast. Nevertheless genetic transformation of lines of soybean and other crop species is a desired objective because of the great agricultural value of the common crop plants and the potential to improve their value and productivity.
In essence, most strategies directed toward the genetic engineering of plant lines involve what generally may be considered two complementary processes. The first process involves the genetic transformation of one or more plant cells of a specifically characterized type. The transformation process is normally defined as introducing a foreign gene, usually a chimeric one, into the genome of the individual plant cells, as typically occurs during Agrobacterium-mediated transformation. The second process involves the regeneration or cultivation of the transformed plant cells into whole sexually competent plants. Neither aspect of the overall strategy is required to be 100 percent successful, or near thereto, but each aspect must have a reasonable degree of reliability and reproducibility so that a practical number of transformed plants can be recovered.
The two processes, transformation and regeneration, must be complementary. It is clearly possible to transform certain tissue or cell types from which the technology does not presently exist to regenerate them into whole plants. For example, it is readily possible, using the technique of electroporation, to readily transform soybean protoplast cells in vitro with foreign genes. However, soybean protoplasts cannot be regenerated. It is also possible to regenerate plant tissues of a number of different tissue and cell type for which no technique has presently been developed for successfully, genetically transforming them. The complementarity of the two halves of this overall procedure must then be such that the tissues which are successfully genetically transformed by the transformation process must be of a type and character and must be in sufficient health, competency and vitality, so that they can be successfully regenerated into a fertile whole plant or successfully used to create germ line plasma so that a whole intact fertile plant containing the foreign gene can be created.
Efforts have been previously made specifically directed to the genetic transformation of soybean cells in culture using Agrobacterium tumefaciens. For example, in Owens et al., Plant Physiology., 77, 87-94 (1985) the responsiveness of soybean cells to A. tumefaciens infections was reported and in Facciotti et al., Bio/Technology, 3, 241-246 (1985) the expression of a chimeric gene in soybean crown gall cultures transformed with A. tumefaciens was reported. Other similar reports suggested that tissues of soybeans can be transformed with oncogenic A. tumefaciens, although it has generally been acknowledged that such tissues are considered to be non-regenerable.
Significant effort has been directed toward the regeneration of soybean plants from various tissue types. Regeneration techniques for plants such as soybeans use as the starting material a variety of tissue or cell types. With soybeans in particular, regeneration processes have been developed that begin with certain differentiated tissue types such as meristems, Cartha et al., Can. J. Bot., 59, 1671-1679 (1981), hypocotyl sections, Cameya et al., Plant Science Letters, 21, 289-294 (1981), and stem node segments, Saka et al., Plant Science Letters, 19, 193-201, (1980) and Cheng et al., Plant Science Letters, 19, 91-99 (1980). There has also been reported the regeneration of whole sexually mature soybean plants from somatic embryos generated from explants of immature soybean embryos Ranch et al., In Vitro Cellular & Developmental Biology, 21: 11, 653-658 (1985). Recent reports also describe the regeneration of mature soybean plants from tissue culture by organogenesis and embryogenesis, Barwale et al., Planta, 167, 473-481 (1986) and Wright et al., Plant Cell Reports, 5, 150-154 (1986).
There has been one report on the use of DNA-coated tungsten projectiles accelerated by a bullet gun to obtain transient expression of foreign DNA in intact epidermal cells of Allium cepa (onion), but no engineered plants have been reported from this method; Klein et al., Nature, 327:70-73 (1987).